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Abstract

Spermatogenesis is an essential stage in human male gamete development, which is regulated by many Y chromosome specific genes. Most of these genes are centred in a specific
region located on the long arm of the human Y chromosome known as the azoospermia
factor region (AZF). Deletion events are common in Y chromosome because of its peculiar structural organization. Astonishingly, among the several known genetic causes of
male infertility, Y chromosomal microdeletions emerged as the most frequent structural
chromosome anomaly associated with the quantitative reduction of sperm. The development of assisted reproductive techniques (ART) like intra-cytoplasmic sperm injection
(ICSI) and testicular sperm extraction (TESE) helps to bypass the natural barriers of
fertilization, but it increases the concern about the transmission of genetic defects. Experimental evidence suggested that the men with Y chromosomal microdeletions vertically transmitted their deletion as well as related fertility disorders to their offspring via
these ART techniques. In India, infertility is on alarming rise. ART centres have opened
up in virtually every state but still most of the infertility centres in India do not choose
to perform Y chromosomal microdeletion diagnosis because of some advanced theoretical reasons. Moreover, there is no consensus among the clinicians about the diagnosis
and management of Y chromosomal microdeletion defects. The current review discusses
thoroughly the role of Y chromosome microdeletion screening in the workup of male
infertility, its significance as a diagnostic test, novel approaches for screening Y deletions
and finally a systematic review on the current status of Y chromosome microdeletion
deletion screening in India.

Introduction

Infertility is a major public health problem with significant
social, psychological and economic impact. In
world literature, there is a paucity of accurate study to
estimate the actual prevalence and incidence of infertility
all around the globe. Noticeably a study carried
out by Boivin et al. (1) estimated that the prevalence
of infertility ranged from 3.5 to 16.7% with an overall
median prevalence of 9% where more than half of
them seek medical care. Worldwide, the incidence of
infertility among the general population is estimated
to be about 10-15% (2).

Infertility is defined as the diminished or absent
ability to conceive or produce an offspring after at
least one year of unprotected sexual intercourse (3).
Being a parent and having a family is the primary vision
of most people in their adulthood. When it ends
in infertility, it brings in enormous emotional trauma,
feelings of sadness, depression and anger. Infertility is indeed a very painful struggle. Traditionally, women
in infertile couples bear the sole responsibility for
the failure to conceive. However, in reality, infertility
is not just limited to women alone. It is identified
that about 50% of infertility are of male origin (4).
In male factor infertility, the human Y chromosome
plays a pivotal role by regulating the male germ cell
development and maintenance (5). The research on Y
specific candidate genes and their relationship with
idiopathic male infertility has been under scrutiny for
several decades now, but sadly, the research is still at
its infant stages and continues to pose vital challenges
for researchers and clinicians alike, but the picture appears
to have become even more challenging with reports
from several groups around the world, that there
are probably several genes or gene sequences within
the target region of the Y chromosome which are deleted.
Furthermore, recent studies have shown that a
significant proportion of men with severe idiopathic
infertility have microdeletions in the Y chromosome
(6). The rapid advancement in assisted reproductive
techniques gives hope to millions of infertile couples
to have their own baby at the same time it raises serious
concerns about the vertical transmission of genetic
defects, including Y chromosomal microdeletions
as well as related fertility disorders to their sons (7).
It implies the necessity for screening Y chromosomal
microdeletions as a routine diagnostic test in the
workup of male infertility.

In India, infertility is on an alarming rise. It opens an
attractive new market for fertility business. Infertility
clinics with assisted reproduction technology (ART)
facilities have opened in virtually every state, both in
rural and urban areas but the quality varies considerably.
Even after several reports emanating from expert
groups in India and abroad about the possibility of
transmission of undetected molecular genetic defects
to the intra-cytoplasmic
sperm injection (ICSI) born
babies, half of the centres do not choose to perform the
advanced genetic screening analysis like Y chromosomal
microdeletion diagnosis for varying reasons.
Denying the benefits of advanced genetic screening
techniques to the ART born babies will adversely
affect their normal and reproductive health. In this
review, we will systematically outline the role of Y
chromosome microdeletion screening in the workup
of male infertility, its significance as a diagnostic test,
novel approaches for screening Y deletions, pros and
cons of currently available techniques and finally to
discuss critically about the current status of Y chromosomal
microdeletion analysis in India.

Genetic aetiology of male infertility

Crossing all the barriers, male infertility is on a rise
than ever before. In about 25-30% of cases the possible
reason for the causes of male infertility is unknown,
and the condition is termed as idiopathic male
infertility. It is believed that genetic factors play the
key role in the aetiology of this condition (8). On analysis
of the semen, the nature of the abnormality can be
identified (Table 1,) (9). Today the advances made in
research regarding the molecular and cellular mechanism
of spermatogenesis helps to characterize many
disorders previously considered as idiopathic. The
most important of them are hypogonadotrophic hypogonadism,
mutations in the androgen receptor, cystic
fibrosis transmembrane conductance regulator gene
mutation, genetic polymorphisms and Y chromosome
linked infertility. Among these, the chromosomal abnormalities
are found much more frequently in infertile
men, with an incidence of 4-16% as compared to
an incidence of 0.4% in the fertile population (10).

The human Y chromosome

The human Y chromosome consists of a short
(Yp) and a long (Yq) arm. Cytogenetic analysis
helped to identify several different Y regions; the
pseudo autosomal portion (including PAR1 and
PAR2), the euchromatic and heterochromatic regions.
The two pseudo autosomal regions (PAR1
and PAR2) located on both telomeric ends cover
around 5% of the chromosome. They are identical
with the appropriate telomeric segments of
X chromosome and the genes localized in PARs
(PAR1 has 14 genes, and PAR2 has 3 genes) show
an autosomal pattern of inheritance (11). Euchromatin
regions consist of Yp and the proximal part
of Yq corresponding to Yq11 with a size of 24
megabases (Mb), while the distal part of the longarm
Yq12 is made of a genetically inert region
called heterochromatin which may vary in length
in different male population. From the evolutionary
point of view, the sex chromosomes (X and Y
chromosomes in the human genome) evolve from
a homologous pair of autosomes (12). Suppression
of recombination between nascent sex chromosomes
endorsed them to evolve independently; it
in turn resulted in the accumulation of male beneficial
genes for the sex determination and spermatogenesis
in a specific region of Y chromosome
called male-specific region of the Y chromosome
(MSY) previously called non-recombining region
of Y (NRY) (11). Instead of the usual recombination
"intra chromosomal gene conversion" or
non-reciprocal transfer of genetic information occurring
between duplicated sequences within the
chromosome take place (13).

Genomic organisation of the male-specific region
of the Y chromosome

The MSY comprises 95% of the chromosome’s
length. The euchromatic sequences of the MSY
belong to three discrete classes; X-transposed, Xdegenerate
and ampliconic. Together these three
sequences constitute around 23 Mb of the chromosome,
including 8 Mb on the short arm (Yp) and
14.5 Mb on the long arm (Yq, 14). The X-transposed
region (combined length of 3.4 Mb) shows
99% similarity to the X chromosome. This can be
acquired through the process of X to Y transposition
that occurred about 3-4 million years ago
(15). X-degenerate sequences (8.6 Mb) represent
the single copy gene or pseudogene homologues
of X-linked genes that appear to be the remnants
of ancient autosomes from which the sex chromosomes
co-evolved. Ampliconic sequences constitute
the major portion of the MSY euchromatic
sequence, where sequence pairs show greater than
99.9% identity organized in massive palindromes.
These amplicons are distributed in seven blocks on
Yp and Yq and whose combined length is 10.2 Mb.
The most outstanding features of the ampliconic
regions of Yq are the presence of eight massive
palindromes. It collectively comprises 5.7 Mb,
or one-quarter of the MSY euchromatin and six
out of eight carrying recognized protein-coding
genes, all of which seem to be expressed specifically
in the testes (14). Among the 156 transcriptional
units located in the euchromatin sequence
of MSY, 78 were protein-coding units encoding at
least 27 distinct proteins or protein families. The
X-degenerate sequences encode 16 distinct proteins,
which were expressed ubiquitously except
the sex determining SRY, which is found to be
expressed predominantly in the testes. The ampliconic
sequences encode 9 of the MSYs 27 distinct
proteins which are specifically expressed in testis
indicating the importance of ampliconic sequence
in Y chromosomal architecture and thereby regulating
spermatogenesis. Ampliconic sequences exhibit
the highest density of genes as well as the
lowest density of interspersed repeat elements.
These sequences comprise sixty coding genes organised
in nine MSY specific protein-coding gene
families and 75 non-coding transcription units; 65
are members of 15 MSY-specific families and the
remaining 10 occur in single copy (14, 16).

Mapping in male-specific region of the Y chromosome

In 1976, Tiepolo and Zuffardi (17) made the
first attempts at mapping the Y chromosome and
thereby proposed a hypothesis that "a gene or
gene cluster on the long arm (Yq11) is essential
for fertility". After a decade, the first molecular
map based on the development of linear deletion
interval maps using Y-specific DNA probes was
developed which subdivided the Y chromosome
into seven deletion intervals (18). Genetic mapping
is impossible in MSY due to the suppression
of meiotic recombination. Therefore, Y chromosome
is the more appropriate target for physical
mapping. Physical maps are based on collections
of overlapping ribosomal DNA (rDNA) clones covering an entire genome. But the early efforts to
construct accurate, high-resolution physical maps
of the MSY were found to be tedious because of
mere availability of specific DNA markers, presence
of large-sized intra-chromosomal repetitive
sequences, or amplicons (19). By using about 200
Y specific sequence tagged sites (STS’s) Vollrath
et al. (20) constructed a 43 interval deletion map
of human Y chromosome, which refined the seven
interval map of Vergnaud et al. (18). These arrays
of STS have been extensively used to build scaffolds
of overlapping recombinant DNA clones.
As a result, the complete physical map of human
Y chromosome was generated with 196 overlapping
DNA clones, which covered 98% of the euchromatic
region (19). This in turn helped with
the sequencing and mapping of MSY using BAC
clones (21). The availability of an MSY reference
sequence has led to the expansion of the current
knowledge of spermatogenesis and Y chromosomal
infertility (14).

Y chromosomal microdeletions

Y chromosome microdeletions (YCM) represent
the absence of DNA segments or gene(s) from
the functionally active part of the Y chromosome.
The first insight into the correlation between the
Y chromosomal microdeletion and male infertility
came from the studies of Tiepolo and Zuffardi
in 1976 (17). Later on with the development of
STS and YAC based mapping, several interstitial
microdeletions which are present on the long arm
of Y chromosome (Yq11) were identified (19-21).
Currently, the Y chromosomal microdeletions assigned
to be the most frequent structural chromosomal
anomaly associated with failure in sperm
production with an overall frequency of 1 to 58%,
specifically 15-20% of idiopathic azoospermic men,
7-10% of idiopathic oligozoospermic men and
2-3% of the candidates for ICSI are carriers of microdeletions
(22). The frequency variation among
various studies is mainly due to the lack of proper
patient selection criteria, ethnic variation among
the study population and differences in the experimental
designs (23).

Azoospermia factor

Besides the factors which control testicular differentiation
and maturation, on the Y chromosome, a third
genetic factor or gene cluster located on the distal portion
of the long arm of the Y chromosome (Yq11 or
deletion interval 5 and 6) controlling the spermatogenesis
is termed as the azoospermia factor (17). It
is said to be the hotspot region of Y chromosomal
microdeletion screening analysis. Detailed molecular
analysis subdivided the azoospermia factor into three
sub-regions, AZFa, AZFb, AZFc along with a fourth
recently proposed AZFd region (24, 25). Each of these
regions comprises functionally active genes and transcription
units related to spermatogenesis. To date, 14
proteins coding genes and two pseudogenes are found
in the AZF locus (26). Partial or complete deletion of
AZF regions impairs spermatogenesis. Several clinically
relevant microdeletion patterns have been identified
in the AZF locus, namely AZFa, P5-proximal P1
(AZFb), P5-distalP1 (AZFbc), P4-distal P1 (AZFbc)
and b2/b4 (AZFc) (27). The deletions are caused by
the intrachromosomal recombination between homologous
sequences (14). The AZF region and the
deletion patterns with corresponding STS markers are
schematically represented in figure 1.

AZFa locus

The AZFa locus lies in the proximal region of
the Yq 11 (D3-D6) or in the deletion, subinterval
5C of the human Y chromosome (25, 28). It
is about 800 kb long and contains two functional
single copy genes, USP9Y and DBY and at least
11 pseudo genes. USP9Y (Ubiquitin specific peptidase
9, Y-linked), which is comprised of 46 exons
and spans 159 kb of genomic DNA whereas, DBY
(Dead box on Y) contains 17 exons and a length
of 16 kb (29). USP9Y gene belongs to a member
of the peptidase C19 family which encodes a protein
which acts like ubiquitin C-terminal hydrolase
and is expressed ubiquitously. The basic function
of USP9Y gene is to increase the efficiency of
spermatogenesis like a "fine-tuner" (30-32). DBY
gene codes for ATP dependent RNA helicases in
humans which play a significant role in pre-meiotic
spermatogonia phase. It has a characteristic
DEAD (Asp-Glu-Ala-Asp) box at the sequence
level and the proteins encoded by the DBY gene
are expressed only in testis tissue (33). Moreover,
mutation events in the candidate genes may lead to
infertility (29, 31).

Mechanism of AZFa deletions

AZFa deletions are low frequency microdeletions
resulting from homologous intrachromosomal
recombination between two human endogenous
retroviral sequences HERV15yq1 and
HERV15yq2 located in the proximal Yq11. The
complete AZFa deletion removes ~792 kb of the
DNA sequences, including the two candidate
genes (34).

Diagnosis of AZFa deletions

Microdeletions in the azoospermia region are
common mutational events. They can be detected
using polymerase chain reaction (PCR) with
the help of STS. The most frequently used gene
specific, single copy STS markers to detect and
study extension of AZFa deletions are DFFRY,
DBY, sY83,sY85, sY86, sY84, sY87, sY88 sY90,
sY1317, sY1316 and sY1234 (25, 35-38).

Clinical significance

The partial removal of AZFa region has been
associated with hypo-spermatogenesis whereas
the complete deletion of AZFa region blocks the
production and maturation of germ cells in the
seminiferous tubule. Consequently, the testicular
biopsy shows the Sertoli cell-only (SCO) phenotype.
It is virtually impossible to retrieve mature
sperm upon testicular sperm extraction (TESE) for
the use in IVF/ICSI (39). Transmission of AZFa
deletion to the offspring is also reported (29).

AZFb locus

AZFb is located in the middle region of Yq11 or
at the deletion interval between P5 palindrome and
the proximal arm of P1 palindrome of the MSY.
It overlaps with the AZFc region by 1.5 Mb and
it spans around 6.2 to 7.7 Mb of MSY sequences
(13). After the identification of the first AZF candidate
gene, RBMY, several single copy genes and
multicopy gene families were observed, some of
them located on the AZFb locus itself and others
which share the AZFc region. The single copy
protein coding genes include EIF1AY (eukaryotic
translation initiation factor 1A, Y-linked), RPS4Y2
(Ribosomal protein S4 Y isoform 2), CYORF15A
(chromosome Y open reading frame 15A), CYORF15B
(chromosome Y open reading frame
15B) and SMCY (Smcy homologue, Y chromosome).
Additionally "seven multicopy gene families
namely XKRY (XK, Kell blood group complex
subunit-related, Y-linked), HSFY (Heat shock transcription
Factor Y), RBMY1A1 (RNA binding motif
protein, Y-linked, family 1, member A1), PRY
(PTPN13-like, Y linked), CDY (Chromodomain
Y), BPY2 (Basic protein Y 2) and DAZ (deleted in
azoospermia)" are present in the AZFb locus (40)
and all the members of these gene families show a
testis specific pattern of expression (41).

Mechanism of AZFb deletions

The homologous recombination between the
Palindrome P5 and the proximal arm of palindrome
P1 of the Yq results in complete AZFb deletions.
Complete P5/proximal-P1 (AZFb) deletion
takes out 6.23 Mb spanning 32 genes and all the
members of the testis-specific gene families present
in the AZFb locus (13).

Clinical significance

Detection of the AZFb deletion has both diagnostic
and prognostic value. Deletions in AZFb
region leads to pre-meiotic spermatogenic arrest
or SCOS and finally results in azoospermia. It is
impossible to recover mature sperm upon TESE
(41, 43).

AZFbc deletions

Deletions which extend from P5 to the distal arm
of P1 (P5/distal-P1 deletions) and from P4 to the distal
arm of P1 (P4/distal-P1 deletions) together constitute
the AZFbc deletions. (Earlier it was thought
to be AZFb+c deletions). AZFbc deletions are considered
to be a significant class of recurrent deletions
in Y-chromosome also representing the largest of the
deletions in the human genome (13). A deletion in
P5/distal-P1 region takes up to 7.66 Mb, including
42 genes or transcripts whereas, P4/distal-P1 deletion
removes 7.03 Mb along with 38 gene copies.

Non-homologous recombination between P5/
distal-P1 or between P4/distal-P1 explains the underlying
mechanism behind AZFbc deletions (13).
On the contrary, one study reported homologous
recombination mechanism for P5/distal-P1 deletions.
The same group identified seven different
types of deletions within AZFb and AZFc regions.
Most of them show non homologous mode of recombination
(44). AZFbc deletions resulted in
impaired spermatogenesis and have no chance of
sperm retrieval through TESE (39).

AZFc locus

The AZFc region is one of the most exhaustively
studied AZF locus associated with male infertility.
AZFc locus is mapped to the distal part of the Yq
or deletion subintervals 6C-6E (25). The AZFc sequence
spans about 4.5 Mb and comprises of six
distinct families of amplicons including six large
inverted repeats and three large direct repeats. Out
of the six inverted repeats three of them are massive
palindromes (P1, P2 and P3) with a size of 4.0
Mb (45). The AZFc locus contains 21 candidate
genes and 11 families of transcription units specifically
expressed in testis. Among the 11 families, 7
families including AZFc-exclusive sequence families,
GOLGA2LY1 (Golgi autoantigen, golginare
subfamily a, 2-like, Y-linked 1) and CSPG4LYP1
(chondroitin sulfate proteoglycan 4-like, Y-linked
pseudogene 1) are specifically located in the AZFc
deletion interval. The important candidate genes in
this deletion interval are four copies of the DAZ
(deleted in azoospermia), three copies of BPY2
(basic protein on Y chromosome 2) and two copies
of CDY1 (CDY1a and CDY1b; chromodomain
protein, Y chromosome 1) gene family (45, 46).
The first identified and well-studied AZFc gene
was DAZ. All the members of DAZ gene family
encode RNA binding proteins, probably involved
in the regulation of mRNA translation, thereby
serving as a vital gene for spermatogenesis (47).

Mechanism of AZFc deletions

AZFc deletions denote the most frequent type of
deletion pattern observed among azoospermic and
severe oligozoospermic patients. The homologous
recombination between sub-amplicons b2 and b4
in palindromes P3 and P1 cause AZFc deletions.
The bordering of the AZFc with the highly repetitive
heterochromatic region of Yq12 may also
trigger a high percentage of unequal intra-chromosomal
recombination during meiotic stage of
spermatogenesis which may increase the chance
of AZFc deletion. The estimated sizes of AZFc deletions
are around 3.5 Mb and result in the elimination
of 21 genes along with seven families of
transcription units, which are solely located within
the deleted region (45).

Diagnosis of AZFc deletions

The STS markers sY254 and sY255, specific
for DAZ gene, are used for the initial screening
of AZFc deletions (47). Extension of the deletion
can be identified using a large set of STS
markers including sY1192, sY1191, sY1291, sY
1035, sY1318, sY254, sY1291, sY1054, sY1190,
sY1263, sY1206 sY 602 (BPY2), sY 1198, sY579,
sY1125 and sY639 (CDY1) (38, 45).

Clinical significance

The patients with AZFc deletions show capricious
phenotypical features, which range from azoospermia to mild/severe oligozoospermia, and
it can apparently be transmitted to the male offspring,
hence the screening of AZFc deletions has a
diagnostic and preventive value. The variations in
the phenotype observed may be due to the genetic
background of the patient screened, exposure to
the environmental factors, size of deletion or 'progression
of spermatogenesis failure with time' (28).
Sperm retrieval from testicular tissue is possible in
AZFc-deleted men when going for ICSI (48).

Partial AZFc deletions and gr/gr deletions

The peculiar structural organization of AZFc region
makes it more prone to large scale structural
rearrangements (45). Recent reports indicate that
several partial AZFc deletions occur in the AZFc
region as a result of recombination between the
sub-amplicons located within the AZFc locus.
Among these gr/gr, b1/b3 and b2/b3 were identified
to be clinically relevant for male infertility
(49). The gr/gr deletions excise 1.6 Mb of the
AZFc region, including four copies of the DAZ
gene and one of three copies of the BPY2 gene.
Since vertical transmission is observed, the gr/gr
deletion likely reduces the fertility of the male offspring
(50). In some men with gr/gr deletions, subsequent
gene duplication helps to restore the gene
copy number (51). The b2/b3 and b1/b3 deletions
also reduce the copy number of the AZFc candidate
genes and alter the normal spermatogenesis
(52). The genotype-phenotype correlation, incidence
and clinical relevance of AZFc partial deletions
are not yet clear.

Genetic screening methods for the diagnosis of Y
chromosomal microdeletions

Novel approaches for screening Y chromosomal
microdeletions

The rapid growth of the molecular diagnostics
helps to introduce a wide array of molecular techniques
to detect the smaller interstitial deletions in
the Y chromosome. Suspension array technology
(SAT) is one among this kind. SAT is based on
flow cytometry principles. It simultaneously analyses
hundreds of molecular targets during a single
reaction. In this technique, the oligonucleotide
probes are allowed to hybridize with microsphere
beads of unique fluorescent label. Once the hybridization
is over the probe hybridized microspheres
are examined using the suspension array analyser.
SAT has been increasingly used to detect the Y
chromosomal microdeletions among the infertile
patients (53). The technique is rapid, specific, sensitive
and cost effective. However, it possesses a
few disadvantages like comparatively low array
size, problems in hybridization and difficulties in
optimizing a single specific annealing temperature
for the entire experiment. Array-comparative
genomic hybridization (CGH) is another powerful
molecular tool used for analysing sub microscopic
Y deletions (54, 55). Another approach for screening
Y deletions is the "use of the capillary electrophoresis
technique combined with fluorescent
multiplex PCR" (56).

The sequence tagged site- polymerase chain reaction
method

The STS-PCR technique is considered to be the
gold-standard method for the laboratory diagnosis
of Y chromosomal microdeletions. In the STSPCR
technique, a DNA sample would be tested for
the presence of STS based on polymerase chain
reaction. The procedure involves many automated
cycles of DNA synthesis in a standard laboratory
thermocycler. Afterwards, the PCR products are
detected with the help of agarose gel electrophoresis.
The presence of the amplified DNA bands indicates
the existence of the known target sequence
and vice versa. The deletion may be a gene or gene
family or may be an unknown sequence depending
upon the STS used (42, 57). While conducting
PCR-based deletion analysis, several questions
have puzzled the mind of clinicians.

Why PCR-based deletion analysis is routinely
used for detecting the Y chromosomal microdeletions?
What factors should be considered while
performing it?

In the early days of research, karyotyping was
commonly used to identify the macrodeletions in
the long arm of Y chromosome. Since the conventional
cytogenetic testing fail to detect the smaller
interstitial deletions, southern blotting took up the
charge to demonstrate the macro as well as the microdeletions
that might be associated in azoo/oligozoospermia
(18). Since karyotyping and southern
blotting are labour-intensive, time consuming,
costly and complex techniques, most laboratories
have switched to PCR which is a simple, reliable,reproducible, less time consuming, cost effective,
sensitive and easily automated technique allowing
multiplexing. Once the PCR based deletion map
was established (20) the search for the interstitial
deletions of the Y chromosome based on PCR
markers began (58), and soon it got accelerated and
hundreds of papers were published within two decades.
The major factors which influence the PCR
based deletion analysis are as follows.

Selection of the polymerase chain reaction markers

The availability of the nucleotide sequence of
the MSY (14) made it possible to select the best
PCR markers from a pool of STS markers (38).
When choosing markers, several factors must be
considered: The finest and informative markers for
the PCR based deletion analysis are single copy
markers or the markers limited to a small region
of the Y chromosome. The gene markers or repetitive
markers like multi copy clustered or dispersed
markers show negative results only when a large
portion of the Y chromosome is deleted, and the
presence of the PCR amplification products will
not indicate all the copies of the target region are
present. Hence the multi copy markers are less informative
(35). The human Y chromosome, as a
hotspot of mutational events, displays structural
polymorphism based on ethnic background and
geographical histories which will in turn reflect on
the PCR markers (35, 59). Therefore, careful selection
of highly specific, non-polymorphic (markers
which are present in fertile men and absent in
infertile men) STS markers is needed to detect the
clinically relevant microdeletion patterns (16, 24).

Origin of the deletions and selection of DNA samples
for screening

Y chromosomal deletions are either inherited
through paternal germ line (7) or occur as de-novo
events. Most of the Y deletion cases are of de novo
origin (7, 28). Most likely, the event takes place
in the pre-fertilization stages, although deletions
could also be a post-fertilization event (60). If a
Y-deleted sperm fuses with an egg, it gives rise to
a Y-deleted child. On the other hand, if the deletion
occurs as a post fertilization event, it will give rise
to mosaicism (normal Y chromosome in leukocytes
and deleted Y chromosome in sperm or testicular
DNA). So the basic question arises at this
point is which DNA sample should be screened for
the detection of Y deletions in the patients opting
for ART? A vast number of studies reported the incidence
of Y chromosomal microdeletions in the
lymphocyte DNA (6, 14, 25). Some of the studies
observed a similar deletion frequency in lymphocyte
and testicular DNA (61, 62). On contrary
few studies found a weak germ cell mosaicism in
oligozoospermic patients (63-65). Unfortunately
these studies have not reported the extent and origin
of the deletions. In one of the studies, evaluating
the relation between sperm DNA damage and
leukocyte DNA integrity, DNA integrity, cleavage
rate and embryo quality were positively correlated
with leucocyte DNA integrity (66). Furthermore,
in azoospermic patients there will be no sperm in
the ejaculate and testicular biopsy collection is still
a highly invasive technique. Also, the collection,
processing and the protocol standardization of
sperm DNA isolation and PCR amplification are
much more complex than the lymphocyte DNA.
Considering all these facts, lymphocyte DNA is
the cheapest and readily available sample for basic
Y chromosomal microdeletion screening.

Polymerase chain reaction quality control

PCR amplification failure leads to the false interpretation
of results. The use of high-quality DNA,
appropriate internal and external positive and negative
controls reduce the false negatives. Moreover,
the European Academy of Andrology (EAA)
guideline for PCR setup and internal quality control
is currently of high value (16).

Reliability of STS markers

STS are short known DNA sequences whose location
in the genome is mapped. The concept of STS
was first put forward by Olson et al. (67). STS offers
high speed, convenient, reliable and low cost genetic
screening analysis. Today about 1287 Y-specific STS,
including 992 single-copy and 285 multi-copy STS
have been generated and mapped to MSY (38). It
was proven that there is no correlation between the
frequency of microdeletions detected and the number
of STS analysed (68). However, for the multiplex
STS-based PCR microdeletion analysis the primary
screening which includes two sets of STS markers in
each AZF sub-region is able to detect over 95% of
the deletions (16). Once deletion has been detected,
secondary screening should be done using 20-30 STS
to detect the extent of deletions (69). If multiple discontinuous deletions are observed the result should be
verified by southern blotting (70).

Patient selection for Y chromosomal microdeletion
screening

Y chromosomal microdeletions are frequently associated
with the quantitative decrease in the sperm
production (27) and also coexist with other male infertility
disorders, testicular cancer and other forms
of human malignancies (71, 72). Furthermore, the Y
deletions are inherited by the offspring; therefore, all
the patients who were the candidates of assisted reproductive
techniques should be also screened for Y
chromosomal microdeletions. If Y chromosomal microdeletions
are observed, subsequent genetic counselling
should be provided to the affected couples.

Growing genetic concerns of assisted reproductive
technologies

The development of ART began with the successful
application of in vitro fertilization (IVF) in
1978. The standard IVF technique was not so effective
for the treatment of people with severe sperm
defects. The introduction of ICSI, the technique by
which an egg is fertilized with the injection of a single
sperm greatly accelerated the practice of ART
globally (73). Since the ICSI technique bypasses all
the natural mechanisms and filters related to normal
fertilization, it raises serious concern about the
transmission of known and unknown molecular
genetic defects to the offspring. Now, the evidence
from various studies shows an increased risk of
congenital malformations and chromosomal aberrations
in children born through ICSI when compared
to the general population. The major risks include
multiple gestations, low birth weight, premature
birth, higher illness morbidity, hearing defects,
genitourinary defects, imprinting defects and chromosomal
aberrations (74-80). Recent reports have
shown an increased risk of gene mutations in the
ART offspring, irrespective of genetic background
(78). It can be speculated that, the occurrence of
cellular damage in the egg during ICSI procedure
increases the incidence of de-novo chromosomal
abnormalities in the developing embryo (81). In addition,
the transmission of Y chromosomal microdeletions,
CFTR gene mutations and DNA repair
defects may possibly affect the health of child born
after ICSI (77, 81). Therefore, proper genetic testing
and counselling should be undertaken to reduce
the genetic risk associated with ART.

Genetic counselling

Genetic counselling is the art of communication between
a professional counsellor and a patient about
a genetic disorder (82). Genetic counselling is mandatory
and now a reality in almost all IVF centres.
Today we have a fair knowledge about the adverse
effects caused by Y chromosomal microdeletions,
and it is also proven to be a potential genetic disorder.
Therefore, genetic counselling should be compulsory
to prevent propagation of this fearful disorder. In one
study, it is reported that most of the couples choose
IVF or ICSI using either the sperm of the partner or
donor sperm only after given proper genetic counselling
about their Yq deletions. In some cases, the couples
choose to select female embryos for transfer (83).
Pre-implantation genetic diagnosis (PGD) seems to
be a potential alternative strategy for the couples dealing
with Yq microdeletions (84).

Y chromosomal screening analysis in India

India, the second most populated country in the
world, exhibits enormous diversity in terms of language,
culture and ethnicity. According to the provisional
reports released on March 31, 2011, the Indian
population has increased to 1.21 billion with a decadal
growth of 17.64%, and a total fertility rate (TFR) of
2.8 children born per woman (85). About 15-20% of
married couples belongs to sub- or infertile category
and a small fraction of these couples opt for ART in
India (86). In the last two decades, several studies
have reported the incidence of Y chromosomal microdeletions
in the Indian population (Table 2,) and
emphasised on the need for the molecular diagnosis
of deletions in the workup of male infertility (87-92).
Even though a large number of infertility clinics are
present in India, half of them still rely on classic cytogenetic
analysis to find out genetic defects. Most of
the infertility centres in India do not choose to perform
Y chromosomal microdeletion diagnosis because of
some advanced theoretical reasons including the test
having no significance in the management of the infertility,
doubts regarding the credibility of diagnostic
techniques, lack of information on genetic counselling
and the variation in the frequencies. Whatever
the reasons, not testing the Y chromosome deletions
is likely to increase the prevalence of complex genetic
diseases associated with Y chromosomal rearrangements
and will deleteriously affect the reproductive
health of the patients and their family.

Table 2 Summary of the literatures on Y chromosomal microdeletion analysis in Indian population

Reference

Region Of Study

Sample Studied

No Of Infertile Men Screened

Control

No Of Sts Markers Used

Frequency Of Deletions

Fertile men

Normozoospermic men

Azoo

Oligo

Others

Total

Ambasudhan et al., 2003

Varanasi

Blood/Testis biopsy

177

?

?

29

8(5.6)%

1(3.3%)

0

9(5%)

Dada et al., 2003

New Delhi

Blood

83

25

0

6

7(9.58%)

1(10%)

0

8(9.6%)

Thangaraj et al., 2003

Kolkata

Blood

340

230

0

37

29(8.5%)

0

0

29(8.5%)

Athalye et al., 2004

Mumbai

Blood

100

5

0

18

8(29.63%)

0

4(5.48%)

12(12%)

Swarna et al., 2004

Hyderabad

Blood

70

?

?

5

4(44.4%)

5(55.5%)

0

9(12.8%)

Rao et al., 2004

South India

Blood

251

?

?

24

5(1.99%)

4(1.59%)

1(0.39%)

10(3.98%)

Dada et al.,2004

New Delhi

Blood

133

50

0

8

7(?)

1(?)

0

8(6.01%)

Dada et al., 2006

New Delhi

Blood

140

50

0

8

?(?)

?(?)

0

8(5.7%)

Mitra et al., 2006

New Delhi

Blood/Semen

14

13

0

19

4 (?)

1 (?)

0

4(28.6%)

Viswambaran et al.,2007

Tamilnadu

Blood

30

20

0

6

? (?)

? (?)

?

4(13.3%)

Saktivel et al., 2008

Tamilnadu

Blood/Semen

147

0

140

34

0

5(7.24%)

14(18.18)

19(12.9%)

Abid et al., 2008

Mumbai

Blood

200

50

0

8

3(3%)

0

3(3%)

6(3%)

Mitra et al., 2008

New Delhi

Blood

170

0

101

19

9(5.29%)

0

0

9(5.29%)

Suganthi et al., 2009

Tamilnadu

Blood

215

?

?

12

? (7.4%)

? (3.7%)

0

24(11.1%)

Pandey et al., 2010

Varanasi

Blood

64

?

?

5

? (?)

?(?)

0

3.33%

Suganthi et al., 2011

Tamilnadu

Blood

100

25

?

12

12(34.29%)

10(25%)

0

22 (29.3%)

The frequency of AZF deletions in the Indian
population

The frequency of Y chromosome microdeletions in
the Indian population ranges from 3 to 29.34 % (87-
93) with an average frequency of 8.1%. It is speculated
that the variation in the frequency of Y deletions
is mainly due to the ethnic background and study protocol.
In one study, a total of 340 azoospermic Indian
men was analysed of which 8.5% showed Y chromosome
deletions, in which AZFc deletion was the most
common (82.8%), followed by AZFb (55.2%) and
AZFa (24.1%, 94). Another study reported the frequency
of Yq microdeletions in the Indian population
as 9.63% (89). A multiplex PCR assay for 18 loci of
the Y-chromosome performed on infertile Indian men
showed that 12% of the patients carry microdeletions,
and the most commonly detected loci were DYS240
and DY6219 (90). Ali et al. (91) have ascertained so far
a total of 109 cases with male infertility from Bangalore
and have shown deletions exist at a frequency of
5.5% in the AZFc region only. In a study by Hellani
et al. (95), a total of 257 patients with idiopathic oligo
or azoospermia were screened for Y-chromosome
microdeletion by typing 19 STS markers in AZF regions.
Among these, six patients had deletions in the AZFc region. One case had a deletion in both AZFa
and AZFc regions. In another study on Indian males,
a total of 215 azoospermic infertile men were tested
for the presence of 12 STS markers using multiplex
PCR. The observed frequency of deletion was about
11.1%, among them the azoospermic men showed a
higher frequency of deletions (7.4%) than the severe
oligozoospermic men (3.7%) (92) [The summary of
the studies on Y chromosomal microdeletion analysis
in the Indian population is shown in table 2]. As
the ethnic and environmental backgrounds affect the
structural arrangements of the Y chromosome, it is
necessary to choose the STS markers carefully for
screening Y chromosome microdeletions based on
ethnic background.

Conclusion

In the era of assisted reproductive techniques,
particularly relating to ICSI, the study of Y
chromosomal microdeletions helps to open up
new horizons. We now have the technology to
test for Y chromosome microdeletions and have
improved knowledge regarding who should be
tested for Yq microdeletions. Additionally, the
Y chromosomal deletion tests have a precise
diagnostic, prognostic and preventive value.
Once a deletion is observed in an infertile man
it helps the clinicians to avoid empirical and
often expensive treatments to improve fertility,
and it also gives information about the chance of
finding sperm in the testes of azoospermic men
and about sperm cryopreservation in oligozoospermic
men. Serious ethical issues may arise
if clinicians promote the desire of couples for
a child without considering the risks involved.
In future, huge demand arises for developing
new molecular technologies as well as standardized
protocols which give reliable results and
also help to increase efficiency, decrease cost
and technical difficulty of the procedure. These
advancements allow a more widespread use of
Y chromosome microdeletion screening test in
infertility clinics and andrology labs.

11Seda O,
Liska F,
Sedova LSex Determination.Multimedia E-textbook of Medical Biology, Genetics and GenomicsCzech RepublicInstitute of Biology and Medical Genetics of the First Faculty of Medicine of Charles University and the General Teaching Hospital2005